EP0037583A1 - Méthode pour la détermination fluorométrique de l'activité des enzymes dégradant les graisses et moyens pour sa réalisation - Google Patents

Méthode pour la détermination fluorométrique de l'activité des enzymes dégradant les graisses et moyens pour sa réalisation Download PDF

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Publication number
EP0037583A1
EP0037583A1 EP81102650A EP81102650A EP0037583A1 EP 0037583 A1 EP0037583 A1 EP 0037583A1 EP 81102650 A EP81102650 A EP 81102650A EP 81102650 A EP81102650 A EP 81102650A EP 0037583 A1 EP0037583 A1 EP 0037583A1
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group
groups
acyl
fluorescent
compound
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EP81102650A
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German (de)
English (en)
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EP0037583B1 (fr
Inventor
Paavo K. J. Kinnunen
Tom M. Schröder
Jorma A. Virtanen
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Ksv-Chemicals Oy
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Ksv-Chemicals Oy
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Priority claimed from FI801117A external-priority patent/FI801117A7/fi
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/61—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving triglycerides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)

Definitions

  • the object of the present invention is a clinico- analytical assay method which is based on changes occurring in the fluorescence intensity.
  • One such method is lipase and phospholipase, especially phospholipase A 2 assay in serum.
  • Lipases are enzymes which split triacyl-glycerols into free fatty acids, glycerides and glycerol. Lipases differ from each other i.a. as to their stereospecificity, and some lipases have the ability to distinguish between the fatty acids in the 1- and 3-positions of an sn-triacyl- glycerol.
  • Phospholipase A 2 splits the fatty acid in the 2-position with respect to the phosphoryl group of a phospholipid, the end products being a lysophospholipid and a free fatty acid.
  • lipase and phospholipase enzymes which under normal conditions are released into the gastrc-intestinal tract. I.a. in connection with pancreatitis these enzymes, however, are released into the blood stream and consequently it is of diagnostical importance to measure the enzyme activity in the plasma.
  • the assay methods for phospholipases have been based on the use of a radioactive substrate, whereby e.g. C- or 3 H -atoms are introduced into the fatty acid in the 2-position and the degree of radioactivity of the fatty acid split by the phospholipase A 2 is measured.
  • the method is however cumbersome because of the many stages involved, and it requires special apparatuses, i.a. a scintillation counter.
  • the object of the present invention is to provide a fluorometric assay method wherein substrates containing glycerol and phosphoglycerol compounds are used, into which compounds fluorescent groups have been introduced, and optionally also quencher groups, and wherein the changes in the fluorescence intensity due to the enzymatic reaction are measured.
  • the object of the present invention is thus a method for fluorometrically measuring the activity of fat-degrading enzymes in samples containing said enzyme according to which method the enzyme containing sample is combined with a substrate which contains an acyl- or an acyl-alkyl-glycerol or -phosphoglycerol which reacts with the enzyme to be assayed, wherein at least one of the acyl or alkyl groups contains a fluorescent group and the other groups may optionally contain a fluorescence quenching group, the substrate is excited at the specific excitation wave-length of the fluorescent group in question and the change, due to the enzyme, in the fluorescence intensity of the substrate per time unit is measured at a specific emission wave length of the fluorescent group, the rate of change being directly proportional to the enzyme activity in the sample.
  • the fluorescent group may be pyrene, tetracene, anthracene, phenanthrene, naphthalene, coumarone, coumarin, acridine, benzocarbazone, aminonaphtalenesulfonic acid, mono-, di- or tri-iodo-benzene, perylene, phenyloxadi- azole, diphenyloxazole, alloxazine, stilbene, dibenzo- furan, fluorene, fluorenone, oxopiperazine, p-quinone, methylumbelliferone, phenazine, phenyl-indole, quinoline, di-ethylaniline, phenol, diphenylacetylene, benzotiophen, pyrimidine, xanthone, thiocarbocyanide, 1,3,5,7-deka- tetra-ene.
  • a suitable fluorescent group is pyrene because of its well-characterized fluorescence behaviour (Th. Forster, Angew. Chem. 81, 364 (1969) and S.C. Charlton et al, The Journal of Biol. Chem. Vol. 251, No 24, 7952 (1976)).
  • the fluorescence may be sensitized when using compounds of the formula I containing two fluorescent groups, by introducing into one of them electron donating groups such as methyl, methoxy, hydroxyl or dimethylamino groups, and into the other electron attracting groups, such as cyano and nitro groups.
  • electron donating groups such as methyl, methoxy, hydroxyl or dimethylamino groups
  • other electron attracting groups such as cyano and nitro groups.
  • halogen such as bromine, iodine or chlorine, or halogen substituted groups, such as halogen substituted phenyl groups.
  • a first sub group a) of the compounds of the formula I comprises for lipase assay suitable triacyl-, diacyl- monoalkyl- and diacyl-glycerols, respectively, wherein at least one of the acyl and alkyl groups is substituted with a fluorescent group, and wherein one other or both the other groups may be substituted with a fluorescence quenching group.
  • triacyl-glycerols or the formula I wherein one, two or all three acyl groups may contain one, two or three fluorescent groups, suitably pyrene groups, as well as 1,3-diacyl-2-alkyl-sn-glycerols, which can be substituted with fluorescent groups as the triacyl-glycerols.
  • Usable intramolecularly quenched compounds of this group are for example triacyl- or l,3-diacyl-2-alkyl-sn- glycerols which in their 2-position contain a fluorescent group, whereby the quenching group, suitably a bromine group, is in the 1- and/or 3-position.
  • the second sub group b) comprises phospholipid compounds suitable for phospholipase A 2 assay and having the formula wherein in the formula Ia the group R 2 and in the formula I b the group R 1 is an afore mentioned acyl group, and wherein at least one of the groups Rand R , or R 1 and R 3 , respectively, contains a fluorescent group, and the other optionally a quenching group.
  • 1,2-diacyl-or l-alkyl-2-acyl-compounds of formula Ia wherein one or both of the groups in the positions 1 and 2 contains a fluorescent group.
  • a suitable compound is also the corresponding compound wherein the fluorescent group has been quenched with a bromine or iodine atom or with some other halogen containing group.
  • a compound which contains a single fluorescent group
  • such a compound forms when emulsified in oil, whereby the individual molecules are packed close together, a so-called intermolecular dimer, i.e. an excimer, which, when excited. at the excitation wave- length of the fluorescent group in question, fluoresces at the excimer wavelength characteristic for this group.
  • the above mentioned compound I 1-oleoyl-2-(4-(3-pyrenyl)-buturoyl)-3-oleoyl-sn-glycerol, forms emulsified in oil, an intermolecular excimer, which, when excited at the excitation wavelength of about 320-345 nm, fluoresces at the excimer wavelength of 470 nm of pyrene.
  • an emulsion reacts with pancreatic lipase there is formed, because the pancreatic lipase does not exhibit stereospecificity, two free fatty acids and 2-(4-(3-pyrenyl)-buturoyl)-glycerol.
  • the change in fluorescence intensity may be followed either at the excimer wavelength of pyrene of ca 470 nm or at its monomer wavelength of ca 390 to 400 nm, and the rate of change of the intensity is directly proportional to the amount of fluorescent compound degraded by the enzyme, i.e..to the enzymatic activity.
  • a strongly fluorescent intramolecular excimer is formed by compounds of the formula I which in the same molecule contain two or three fluorescent groups. As a result of the enzymatic reaction the interaction between these fluorescent groups gradually disappears, whereby the excimer fluorescence intensity decreases and correspondingly.the monomer fluorescence intensity increases.
  • the phospholipase A 2 hydrolyzes the fatty acid chain in the 2-position and as reaction products a fluorescent free fatty acid and a fluorescent pyrene-fatty acid phosphatide are formed.
  • the pyrene excimer fluorescence at the wavelength of about 470 nm weakens and its monomer fluorescence at the wave- length of about 400 nm increases, and the rate of change of the fluorescence intensity is proportional to the degree of hydrolyzis.
  • the rate of change of the fluorescence intensity it is possible to determine the amount of fluorescent compound degraded per time unit, which in turn is dependent on the enzymatic activity.
  • the enzymatic activity can be measured also by using according to the invention a compound which contains both a fluorescent as well as a group, or groups, preferably bromine atoms, quenching the intramolecular fluorescence.
  • a fatty acid containing either a fluorescent group or a quenching group is split off and a fluorescent fatty acid or a fluorescent glycerol compound is formed which fluoresces at the monomer wavelength.
  • the increase in intensity, due to a lesser degree of quenching, is enhanced by the same phenomenon as when using a compound containing only one fluorescent group.
  • the method may be carried out also by using an oil-emulsified substrate containing both a compound having one fluorescent group and a compound which contains a fluorescence quenching group.
  • oil these compounds are forced into close contact thus forming a so-called intramolecularly quenched macromolecule, which as a result of the enzymatic reaction is broken down into products which fluoresce at the monomer wavelength.
  • An object of the invention are also the compounds usable for carrying out the method according to the invention, especially the compounds of formula I, as new substances.
  • These compounds may be prepared e.g. by introducing into glycerol or D-mannitol the desired acyl and alkyl substituents, optionally containing fluorescent or quencher groups. The substituted D-mannitol is then split and reduced to the corresponding glycerol. A substituted glycerol thus obtained may be further substituted in its free position with a phosphoryl group or a derivative thereof to obtain the desired compound.
  • Phospholipase A 2 activity was measured using the following phospholipid substrate which in 2.90 ml contains
  • the fluorescence was measured with a Perkin-Elmer-fluorescense- spektrofotometer, excitation opening 3 nm, emission opening 6 nm, excitation wavelength 320 nm, emission wavelength 470 nm.
  • the base level was determined whereafter 100 ⁇ l of a sample containing phospholipase A 2 from cobra venom was added to the substrate.
  • the lipase activity was measured using the following lipid substrate
  • the fluorescence was measured using a fluorescence spectrofotometer connected to a recorder, excitation opening 20 nm, emission opening 20 nm, excitation wave- length 343 nm. Emission was folled at the wavelength 400 nm. First the base level was measured whereafter 50 ⁇ l of a sample containing lipase was added and the increase in fluorescence intensity per time unit was followed at the monomer wavelength of ca 400 nm.
  • Fig. 2 is shown an assay with normal and pathological serum.
  • the fluorescence intensity Im was measured at the monomer wavelength 400 nm as a function of time.
  • fluorescent compound the above 1-oleoyl-2-(4-(3-pyrenyl)-buturoyl-3-oleoyl-sn-glycerol was used. Benzene boronate was used for stopping the a reaction.
  • Example 2 may be repeated but using as an intramolecularly quenched compound 1 ⁇ g of 1-(6-bromohexanoyl) - 2-(4-(3-pyrenyl)-buturoyl)-3-oleoyl-sn-glycerol and measuring the increase in fluorescence intensity per time unit, as above.
  • the phospholipase A 2 activity was measured using a substrate which in 2.0 ml contains
  • phospholipids As phospholipids the following compounds may be used:
  • the lecithin and the fluorescent compound were dried solvent free in a nitrogen stream. Thereafter the Na-deoxycholate was added and the mixture sonicated using a Branson sonifier equipped with microtip at setting 4. Thereafter the bovine serum albumin was added, dissolved in buffer. The substrate was stable for several day provided 0.1 mM NaN 3 was added to prevent microbial growth.
  • Fluorescence changes were measured using a Kontron SFM-23 spectrofluorometer equipped with a magnetically stirred cell (1.0 x 1.0 x 0.5 cm). Temperature was controlled with a cryostat at 37 0 C throughout the measurement. The fluorescence intensity signal was fed into a recorder. Excitation wavelength was 343 nm for the pyrenyl containing compounds and 370 nm for the anthroyl containing compounds, and the changes in the monomer fluorescence intensity were followed 400 nm for pyrenyl and at 450 nm for anthroyl.
  • the lipase activity was measured using a substrate which in 2.0 ml contains
  • the lipid to be tested was dried in a stream of nitrogen whereafter the tributyrin and detergents were added, as well as the buffer solution, whereafter the mixture was sonified as in Example 3.
  • the sample a was to be stored 0.1 mM of NaN 3 was added to prevent microbial growth.
  • the substrate was stable for several days. The solution is to be thoroughly stirred prior to use.
  • Fig. 1 Degradation of radioactive (A) and fluorescent (B) phospholipid in a phospholipase A 2 reaction.
  • Enz. unit enzymatic activity, nanomoles lysophospholipid per minute and ml.
  • the amount of enzyme used was varied and the enzymatic activity measured with both methods.

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  • Organic Chemistry (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
EP81102650A 1980-04-09 1981-04-08 Méthode pour la détermination fluorométrique de l'activité des enzymes dégradant les graisses et moyens pour sa réalisation Expired EP0037583B1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT81102650T ATE10112T1 (de) 1980-04-09 1981-04-08 Verfahren zur fluorimetrischen bestimmung der aktivitaet von fettspaltenden enzymen und mittel zur durchfuehrung dieses verfahrens.

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
FI801117A FI801117A7 (fi) 1980-04-09 1980-04-09 Rasvoja hajoittavien entsyymien aktiivisuuden määritys fluorometrisesti.
FI801117 1980-04-09
FI801258 1980-04-18
FI801258 1980-04-18
FI810616 1981-02-27
FI810616A FI63064C (fi) 1980-04-09 1981-02-27 Foerfarande foer fluorometrisk bestaemning av aktiviteten av fettspjaelkande enzymer och medel foer att genomfoera foerfarandet

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EP0037583A1 true EP0037583A1 (fr) 1981-10-14
EP0037583B1 EP0037583B1 (fr) 1984-10-31

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US (1) US4668623A (fr)
EP (1) EP0037583B1 (fr)
CA (1) CA1154453A (fr)
DE (1) DE3166904D1 (fr)
FI (1) FI63064C (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3342106A1 (de) * 1982-11-19 1984-06-14 Toyo Jozo K.K., Tagata, Shizuoka Komposition fuer lipase-bestimmung
EP0489839A4 (en) * 1989-08-29 1992-10-28 The Regents Of The University Of California Novel hydrolytic enzyme inhibitors and substrates and assays, methods and kits embodying same
US5308766A (en) * 1989-08-29 1994-05-03 The Regents Of The University Of California Hydrolytic enzyme inhibitors/inactivators and methods for using same
US5352673A (en) * 1989-08-29 1994-10-04 Dennis Edward A Prodrugs
WO1995032181A1 (fr) * 1994-05-19 1995-11-30 Progen Biotechnik Gmbh Determination par fluorescence de l'activite d'enzymes lipolytiques
GB2308189A (en) * 1995-12-13 1997-06-18 Univ Sunderland Monitoring an enzyme involving a substrate therefor labelled with a fluorophore
WO2003069305A3 (fr) * 2002-02-13 2004-04-01 Wisconsin Alumni Res Found Dosage de phospholipase fluorescent, inhibiteur et stimulateur de phospholipase a2, et utilisations
WO2003080554A3 (fr) * 2002-03-27 2004-04-15 Fuji Photo Film Co Ltd Derive d'ester de glycerol
US7968306B2 (en) 2002-02-13 2011-06-28 Wisconsin Alumni Research Foundation Method for measuring activity of a specific fraction of albumin

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3614647A1 (de) * 1986-04-30 1987-11-05 Euratom 7-phenylessigsaeure-4-alkyl-coumarinylamide, verfahren zu ihrer herstellung sowie ihre verwendung in verfahren zur fluorometrischen bestimmung der aktivitaet von hydrolasen, insbesondere von penicillin-g-acylase
WO1990000618A1 (fr) * 1988-07-08 1990-01-25 Jbl Scientific, Inc. Preparation et utilisation de derives de benzothiazole fluorescents
US5567596A (en) * 1994-12-29 1996-10-22 Research Foundation Of State University Of New York Rapid assay of activators and inhibitors of clotting
US5908751A (en) * 1996-04-26 1999-06-01 Toyo Ink Mfg. Co., Ltd. Method for detecting and/or determining ATP from microorganism cells in a sample
US6066446A (en) * 1997-12-19 2000-05-23 Nen Life Science Products, Inc. Assay member and method for its manufacture
US5972595A (en) * 1997-12-19 1999-10-26 Nen Life Science Products, Inc. Enzyme assay using a solid phase substrate
DE19919634C2 (de) * 1999-04-30 2001-06-07 Aventis Pharma Gmbh Test zur Bestimmung der Integrität komplexer Phospholipid/Lipid-Strukturen mit Hilfe synthetischer fluoreszenz-markierter Acylglyceride und dessen Anwendung zur Bestimmung der Aktivität von Lipasen
JP4111669B2 (ja) 1999-11-30 2008-07-02 シャープ株式会社 シート製造方法、シートおよび太陽電池
WO2005005977A2 (fr) * 2003-06-30 2005-01-20 Applera Corporation Dosages de phospholipases fluorescentes et compositions
US20050239217A1 (en) * 2003-11-26 2005-10-27 Applera Corporation Fluorogenic homogeneous binding assay methods and compositions
CA2548407A1 (fr) * 2003-12-12 2005-07-07 Qtl Biosystems Llc Essais, kits et reactifs de superextinction de fluorescence induite par des ions metalliques
WO2007012100A1 (fr) * 2005-07-27 2007-02-01 Technische Universität Graz Phospholipides oxydes comportant un fragment de fluorophore et leur utilisation pour determiner la presence d'une enzyme a activite antiatherogene
RU2517746C1 (ru) * 2012-11-22 2014-05-27 Федеральное государственное бюджетное учреждение науки институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наук (ИБХ РАН) Флуоресцентный зонд и тест-система для определения активности фосфолипазы а2
CN103242474B (zh) * 2013-05-08 2015-04-29 南京大学 磷酰胆碱结构修饰的苝酰亚胺衍生物及其制备方法

Citations (3)

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FR7600M (fr) * 1967-04-14 1970-01-19
US3741876A (en) * 1970-08-06 1973-06-26 Mason Res Inst Inc Method of measuring enzyme reaction rates
US3986930A (en) * 1974-05-28 1976-10-19 Dainippon Pharmaceutical Co., Ltd. Lipase activity determining method and reagent

Family Cites Families (2)

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US4261968A (en) * 1979-05-10 1981-04-14 Syva Company Fluorescence quenching with immunological pairs in immunoassays
FI60700C (fi) * 1980-04-09 1982-03-10 Ksv Chemicals Oy Foerfarande foer framstaellning av 1,2-diacyl-sn-glyceroler

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
FR7600M (fr) * 1967-04-14 1970-01-19
US3741876A (en) * 1970-08-06 1973-06-26 Mason Res Inst Inc Method of measuring enzyme reaction rates
US3986930A (en) * 1974-05-28 1976-10-19 Dainippon Pharmaceutical Co., Ltd. Lipase activity determining method and reagent

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 70, no. 15, April 14, 1969, page 28, abstract 64514y, COLUMBUS, OHIO (US) J.G. MEYER-BERTENRATH et al.: "Properties of new substrates for the determination of pancreas enzymes" & Z. Klin. Chem. Klin. Biochem., 6(6), 1968, 484-488 *
CHEMICAL ABSTRACTS, vol. 75, no. 17, October 25, 1971, page 27, abstract 105296c, COLUMBUS, OHIO (US) M. FLEISHER et al.: "Automated, fluorometric procedure for determining serum lipase" & Clin. Chem. 17(5), 1971, 417-422 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3342106A1 (de) * 1982-11-19 1984-06-14 Toyo Jozo K.K., Tagata, Shizuoka Komposition fuer lipase-bestimmung
EP0489839A4 (en) * 1989-08-29 1992-10-28 The Regents Of The University Of California Novel hydrolytic enzyme inhibitors and substrates and assays, methods and kits embodying same
US5308766A (en) * 1989-08-29 1994-05-03 The Regents Of The University Of California Hydrolytic enzyme inhibitors/inactivators and methods for using same
US5352673A (en) * 1989-08-29 1994-10-04 Dennis Edward A Prodrugs
US5427919A (en) * 1989-08-29 1995-06-27 The Regents Of The University Of California Hydrolytic enzyme inhibitors/inactivators and methods for using same
WO1995032181A1 (fr) * 1994-05-19 1995-11-30 Progen Biotechnik Gmbh Determination par fluorescence de l'activite d'enzymes lipolytiques
GB2308189A (en) * 1995-12-13 1997-06-18 Univ Sunderland Monitoring an enzyme involving a substrate therefor labelled with a fluorophore
GB2308189B (en) * 1995-12-13 1999-08-11 Univ Sunderland Method for monitoring enzymes
WO2003069305A3 (fr) * 2002-02-13 2004-04-01 Wisconsin Alumni Res Found Dosage de phospholipase fluorescent, inhibiteur et stimulateur de phospholipase a2, et utilisations
US7579156B2 (en) 2002-02-13 2009-08-25 Wisconsin Alumni Research Foundation Fluorescent phospholipase assay, phospholipase A2 inhibitor and stimulator, and the use thereof
US7968306B2 (en) 2002-02-13 2011-06-28 Wisconsin Alumni Research Foundation Method for measuring activity of a specific fraction of albumin
WO2003080554A3 (fr) * 2002-03-27 2004-04-15 Fuji Photo Film Co Ltd Derive d'ester de glycerol
US7371877B2 (en) 2002-03-27 2008-05-13 Fujifilm Corporation Glycerol ester derivative

Also Published As

Publication number Publication date
EP0037583B1 (fr) 1984-10-31
DE3166904D1 (en) 1984-12-06
FI63064B (fi) 1982-12-31
CA1154453A (fr) 1983-09-27
US4668623A (en) 1987-05-26
FI810616L (fi) 1981-10-10
FI63064C (fi) 1983-04-11

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